A putative RNA-interference-based immune system in prokaryotes: computational analysis 
of the predicted enzymatic machinery and hypothetical mechanisms of action 

Kira S. Makarova

All archaeal and many bacterial genomes contain Clustered Regularly Interspaced Short Palindrome Repeats (CRISPR)
 and variable arrays of the CRISPR-associated (cas) genes. The protein sequences of the numerous cas gene 
products were classified into ~25 distinct protein families; several new functional and structural predictions 
are described. Comparative-genomic analysis of CRISPR and cas genes leads to the hypothesis that the function 
of the CRISPR-Cas system (CASS) is defense against invading phages and plasmids via a mechanism analogous 
to the eukaryotic RNA interference (RNAi). Specific functional analogies are drawn between several components 
of CASS and proteins involved in eukaryotic RNAi, including the double-stranded RNA-specific helicase-nuclease 
(dicer), the endonuclease cleaving target mRNAs (slicer), and the RNA-dependent RNA polymerase. 
However, none of the CASS components is orthologous to its apparent eukaryotic functional counterpart. 
It is proposed that unique inserts of CRISPR, some of which are homologous to fragments of bacteriophage 
and plasmid genes, function as prokaryotic siRNAs (psiRNA), by base-pairing with the target mRNAs and promoting 
their degradation or translation shutdown. Specific hypothetical schemes are developed for the functioning 
of the predicted prokaryotic siRNA system and for the formation of new CRISPR units with unique inserts 
encoding psiRNA conferring immunity to the respective newly encountered phages or plasmids. 
The unique inserts in CRISPR show virtually no similarity even between closely related bacterial strains 
which suggests their rapid turnover, on evolutionary scale. Taken together, these findings indicate that, 
most likely, CASS is a prokaryotic system of defense against phages and plasmids that functions via the 
RNAi mechanism.

Joint work with Yuri Wolf, Nick Grishin, Svetlana Shabalina, and Eugene Koonin